Nasal spray formulation and method

ABSTRACT

A nasal spray formulation for use in female contraception or in the treatment of benign gynecological disorders is described. The nasal preparation is comprised of a GnRH compound and an estrogenic compound in the form of a water-soluble complex with a water-soluble cyclodextrin. The preparation effectively suppresses ovarian estrogen and progesterone production, and prevents signs and symptoms of estrogen deficiency, without a significant increase in the risk of endometrial hyperplasia.

This application is a continuation of U.S. application Ser. No.10/298,378, filed Nov. 15, 2002, now pending; which claims the benefitof U.S. Provisional Application No. 60/400,575, filed Aug. 2, 2002, nowabandoned, both of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to compositions comprising a gonadotropinreleasing hormone compound and an estrogenic compound for mucosaldelivery, particularly nasal delivery, for contraception and/ortreatment of benign gynecological disorders. The invention also relatesto methods for preparing the compositions and to methods of treatmentusing such compositions.

BACKGROUND OF THE INVENTION

During a woman's reproductive life, a delicate and complex interplay ofhormones are timed and controlled by the hypothalamus. The hormones thatparticipate in the feedback system regulating the menstrual cycleinclude estrogens and progesterone, the pituitary gonadotropins FSH(follicle stimulating hormone) and LH (luteinizing hormone), andgonadotropin-releasing hormone (GnRH) from the hypothalamus.

Manipulation of the hormonal balance is a recognized approach ofcontraception and of treatment of benign gynecological disorders. Inparticular, administration of a GnRH compound for contraception has beendescribed (U.S. Pat. Nos. 5,340,584; 5,211,952). Typically, the GnRHcompound is administered in a slow or controlled-release fashion forcontinuous suppression of ovarian estrogen and progesterone production.Estrogen, often a progestin, and sometimes an androgen, are “added-back”to ameliorate the effects of hormonal deficiency. The hormone add-backsare also often administered in a slow, controlled-release ortime-release fashion to maintain a constant hormonal serum level.

Treatment of benign gynecological disorders by administration of a GnRHcompound has also been described (U.S. Pat. Nos. 5,340,584; 5,340,585;5,681,817). During a women's reproductive years, defined as the timebetween onset of menses (menarche) and the final episode of bleeding(menopause), that is a premenopausal woman, a variety of benigngynecological disorders can occur. Common benign gynecological disordersinclude, but are not limited to, premenstrual syndrome, endometriosis,uterine leiomyomata (uterine fibroids), and polycystic ovarian syndrome.As for contraceptive use, a GnRH compound is administered to suppressovarian follicle development and sex steroid production to relieve ortreat symptoms associated with the disorder.

The administration of drugs by absorption through mucosae, such as thenasal mucosa or vaginal mucosa, has been of considerable interest inrecent years. This route of drug delivery is an alternative to oraladministration in cases where drugs are poorly absorbed or areextensively metabolized in the gastrointestinal tract or subjected tofirst-pass metabolism in the liver. In particular, nasal mucosa has thedesirable properties of being highly vascular leading to rapid uptakeand the avoidance of first-pass metabolism in the liver, since thevenous system from the nose passes directly into the systemiccirculatory system. The nasal mucosa also exhibits moderate permeabilityto water-soluble compounds, comparable to that of the ileum. Thepermeability of nasal mucosa is higher for most compounds than othermucosae, due in part to the difference in structure of the cells liningthe body cavities.

Efficiency of delivery of drugs by an intra-nasal route is influenced bythe degree and rapidity of enzymatic degradation, the nasal clearancerate, as well as the drug's permeability through the mucosa. Theclearance rate is produced by the coordinated movement of cilia and isknown to be highly dependent upon the prevailing physiological andpathological conditions. Thus, for many drugs administrationintranasally is inefficient due to low uptake of the drug, hence lowbio-availability.

Another potential problem associated with intranasal delivery is mucosalirritation. Irritation caused by the drug itself and/or by absorption orpenetration promoters or enhancers often limits the success of nasalformulations. Chronic administration of irritating nasal formulationscan cause necrosis, inflammation, exudation, removal of the epithelialmonolayer or can lead to irreversible damage to the nasal mucosa.

Nasal formulations comprised of a GnRH compound have been described(see, for example, U.S. Pat. Nos. 5,116,817; 4,476,116). However, it isunknown if intranasal delivery of a composition containing multipleactive agents, such as a GnRH compound and one or more hormonal agents,is suitable for contraception or for treatment of benign gynecologicaldisorders. For example, it is unknown if the presence of multiple agentsin the formulation interfere with absorption of one or another of theagents. Formulations comprised of a GnRH compound and one or morehormonal agents that are sufficiently non-irritating to the nasal mucosafor commercial viability have also not been described.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a nasalpreparation having a GnRH compound and an estrogenic compound, andoptionally an androgenic compound, where uptake of the GnRH compound inthe presence of the estrogenic and optional androgenic compounds issufficient for therapeutic activity; i.e., uptake of the GnRH compoundis substantially unhindered by the steroids.

It is another object of the invention to provide a bolus-form ofdelivery of a composition comprised of a GnRH compound and an estrogeniccompound, and optionally an androgenic compound, that offers atherapeutic activity similar to that of a slow-release composition ofthe same active agents, with similar hormonal areas under the curve.

In one aspect, the invention includes a nasal spray formulation for usein female contraception or in the treatment of benign gynecologicaldisorders, the composition comprising an aqueous medium having dissolvedtherein (i) a GnRH compound and (ii) an estrogenic compound present inthe form of a water-soluble complex with a water-soluble cyclodextrin.The concentration of GnRH compound and estrogenic compound in theformulation are effective, when administered daily in the form of aliquid aerosol having a total liquid volume between 30 and 200 μL, andover an extended period of administration, to suppress ovarian estrogenand progesterone production and to prevent signs and symptoms ofestrogen deficiency, without a significant increase in the risk ofendometrial hyperplasia.

The GnRH compound can be an agonist or an antagonist, and exemplarycompounds include deslorelin, leuprolide, nafarelin, goserelin,decapeptyl, buserelin, histrelin, gonadorelin, abarelix, cetrorelix,azaline B, and degarelix, and analogs thereof.

In one embodiment, the cyclodextrin is 2-hydroxypropyl-β-cyclodextrinand is present in the formulation at a concentration between 50 and 300mg/mL. In another embodiment, the 2-hydroxypropyl-β-cyclodextrin has adegree of substitution between 2 and 8, more preferably between 5 and 8.

In a preferred embodiment, the GnRH compound is deslorelin, at a dailydose between 0.025 and 1.5 mg.

In another preferred embodiment, the estrogenic compound is17-β-estradiol, at a daily dose between 0.15 and 0.6 mg.

In another embodiment, the formulation further includes testosterone asa second or third steroid in the form of a water-soluble complex withthe cyclodextrin, and at a daily dose of between 0.15 and 1 mg.

In still another embodiment, the formulation further includes aprogestin as a second or third steroid in the form of a water-solublecomplex with the cyclodextrin.

The estrogenic compound and the second and or third steroid can have acombined molar occupancy with respect to the cyclodextrin that isgreater than the molar occupancy achievable with any of the steroidsalone.

In another aspect, the nasal preparation described above whenintranasally administered as a daily bolus (i) is effective to achievean average serum concentration over 24 hours of the estrogenic compoundthat is within 10% of the average serum concentration over 24 hours ofthe estrogenic compound when delivered transdermally and (ii) is aseffective in preventing bone mineral density loss as transdermaladministration of the estrogenic compound.

In another aspect, the invention includes an intranasal drug-deliverysystem for use in female contraception or in the treatment of benigngynecological disorders. The system is comprised of a nebulizer operableto deliver a selected volume between 30 and 200 μL of an aqueousformulation in the form of a liquid-droplet aerosol. Contained in thenebulizer is a liquid formulation composed of (i) a liquid carrier, (ii)a GnRH compound capable of suppressing ovarian estrogen and progesteroneproduction, and (iii) an estrogenic compound capable of preventing signsand symptoms of estrogen deficiency when co-administered with the GnRHcompound. The concentration of GnRH compound and estrogenic compound inthe formulation are effective, when administered once daily in the formof a liquid aerosol having a total liquid volume between 30 and 200 μL,and over an extended period of administration, to suppress ovarianestrogen and progesterone production and to prevent signs and symptomsof estrogen deficiency, without a significant increase in the risk ofendometrial hyperplasia.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying examples and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the molar ratio of steroid to2-hydroxypropyl-β-cyclodextrin for testosterone (T/HPβCD), for estradiol(E2/HPβCD), and for testosterone and estradiol in combination where thetwo steroids are added to the 2-hydroxypropyl-β-cyclodextrin solutionsimultaneously (T+E2/HPβCD) or added sequentially (E2 then T/HPβCD).

FIGS. 2A-2B are bar graphs showing the baseline corrected cumulativearea under the curve from 0 to 360 minutes of serum estradiol inpg·min/mL (FIG. 2A) and of serum testosterone in ng·min/dL (FIG. 2B) forsix patients treated with an intranasal preparation comprised of theGnRH compound deslorelin, estradiol, and testosterone. The preparationwas administered twice (days 1 and 8), one week apart.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

An “extended time period” intends a period of more than about 4 months,preferably more than about 6 months.

The phrase “amount effective to suppress ovarian estrogen andprogesterone production” refers to a dose of a therapeutic compound, andin particular to a dose of a GnRH compound, that reduces serum estrogenlevels such that one or more symptoms typically associated withmenopause, such as vasomotor instability, bone loss, decreased libido,vaginal dryness, and/or urogenital atrophy, are observed. Typically, asustained serum estrogen level of less than about 30 pg/mL, moretypically of less than about 20 pg/mL, is considered as evidence thatovarian estrogen production is suppressed.

The phrase “amount effective to suppress ovarian progesteroneproduction” refers to a dose of a therapeutic compound, and inparticular to a dose of a GnRH compound, that maintains serumprogesterone at a level consistent with anovulation. Typically, asustained serum progesterone level of less than about 80 ng/dL, moretypically of less than about 50 ng/dL, is considered as evidence thatovarian progesterone production is suppressed.

The phrase “amount effective to prevent signs and symptoms of estrogendeficiency” refers to a dose of a therapeutic compound that inhibits orminimizes clinically-recognized markers of estrogen deficiency,including but not limited to symptoms typically associated withmenopause, such as vasomotor instability, bone loss, and/or urogenitalatrophy.

The phrase “without significant risk of endometrial hyperplasia” intendsa risk of developing simple endometrial hyperplasia that is less thanthe average risk of a given population of women. For example, theaverage risk of hyperplasia after one year of treatment with unopposedestrogen in the general population of postmenopausal (naturally orsurgically menopausal) women treated with unopposed estrogen (0.625 mgdose) is about 30% (Gefland, M. et al. Obstetrics & Gynecology, 74:398(1989)). Thus, ‘no significant risk of endometrial hyperplasia’, withrespect to postmenopausal women treated with 0.625 mg daily unopposedestrogen, would be less than 30%, more preferably less than 20%, stillmore preferably less than 10%, even still more preferably less than 5%,of a test population developing simple endometrial hyperplasia.

The term “GnRH compound” as used herein intends peptide and non-peptideGnRH analogs, and includes agonists and antagonists. Exemplarynon-peptide analogs are described, for example, in U.S. Pat. No.6,346,534, and these exemplary non-peptide analogs are incorporated byreference herein. Peptide analogs are widely reported in the literature.

The terms “progestin” and “progestogen” are used interchangeably.

II. NASAL FORMULATION

As noted above, the invention includes a nasal preparation for use infemale contraception or in the treatment of benign gynecologicaldisorders. In this section, each component in the composition will bedescribed.

A1. Composition Components: GnRH Compound

The composition for use in the method of the invention comprises a GnRHcompound. Native GnRH is a decapeptide comprised of naturally-occurringamino acids having the L-configuration, except for the achiral aminoacid glycine. The sequence of GnRH is(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (SEQ ID NO:1). A largenumber of analogs of this natural peptide have been prepared and areeffective to inhibit the release and/or the action of GnRH. Analogshaving agonist and antagonist activity have been described, and as usedherein, the term “a GnRH compound” or “GnRH compounds” intends agonistsand antagonists, synthetically prepared or naturally-occurring, peptideand non-peptide compounds alike. The following description focuses inparticular on GnRH agonists, however, it will be appreciated that nativeGnRH, GnRH antagonists, such as azaline B, abarelix, cetrorelix, anddegarelix, and other GnRH analogs are also suitable for use in thecomposition and method of treatment. Further, the following discussionfocuses on peptide analogs, however, it will be appreciated thatnon-peptide compounds, such as those disclosed in U.S. Pat. No.6,346,534, are also contemplated.

GnRH agonists are compounds that work in two phases. The first phasestimulates the ovaries to produce more estradiol. During the secondphase, the messenger hormones that control the ovaries are suppressed,resulting in a drop in estrogen. An exemplary agonist is obtained bychanging the 6-position residue in the naturally-occurring GnRH from Glyto a D-amino acid, to give a GnRH analog having a sequence(pyro)Glu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Gly-NH₂ (SEQ ID NO:2), where Xrepresents an amino acid in the D-configuration. When X is D-Leu theanalog is known as Lupron™ and is commercially available from TAPPharmaceuticals (Lake Forest, Ill.). Agonists often have the N-terminusprolyl modified with an n-ethylamide addition. For example, the agonistdeslorelin is (pyro)Pro-His-Trp-Ser-Tyr-DTrp-Leu-Arg-Pro-ethylamide (SEQID NO:3). Another exemplary analog is where the 6-position residue isD-Ala to give a peptide having the following sequence:(pyro)Glu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH₂ (SEQ ID NO:4; U.S.Pat. No. 4,072,668). Another exemplary agonist is obtained byeliminating the Gly-NH₂ in position 10 to give a nonapeptide as analkyl, cycloalkyl, or fluoroalkylamide, or by replacing Gly-NH₂ by anα-azaglycine amide. Modifications to the naturally-occurring GnRHsequence at positions 1 and 2 are also possible. A number of GnRHagonists are described in the art, many of which are commerciallyavailable, and include deslorelin, leuprolide, nafarelin, goserelin,decapeptyl, buserelin, histrelin, and gonadorelin, and analogs thereof.

The dose of the GnRH compound is preferably sufficient to suppressovarian estrogen and progesterone production, so that estrogen effectsare predictably related to the co-administered estrogenic compound,described below. The amount of GnRH compound effective to achieve thedesired suppression of ovarian estrogen production may readily bedetermined with respect to any given GnRH compound and for any givenmammal. The dose range depends upon the particular GnRH compound usedand may also depend upon patient characteristics, such as age andweight. Further, the effective amount of GnRH compound also depends uponroute of administration. Determination of an effective dose range afterconsideration of these factors is routine for those of skill in the art.

By way of example of a specific formulation, the amount of GnRH compoundin a daily nasal spray formulation with a volume between about 30 toabout 200 μL can deliver a daily dose of GnRH compound of between about0.025 mg to about 1.5 mg. It will be appreciated that the daily sprayvolume can be administered in one, two, or more separate deliveries toachieve the desired total daily spray volume. It will further beappreciated that the spray volume and the amount of GnRH compound in thenasal formulation are each individually adjustable to achieve thedesired daily dosage.

A2. Composition Components: Estrogenic Compound

A second component in the composition for use in the method of theinvention is an effective amount of an estrogenic compound. Theestrogenic compound is effective to prevent symptoms and signs ofestrogen deficiency including bone loss, vaginal atrophy, and hotflashes.

The estrogenic compound can be a single-component natural or syntheticestrogen composition, or a combination of such compounds. As usedherein, the term “estrogenic compound” refers to both natural andsynthetic materials having activity to mitigate the signs and symptomsof estrogen deficiency. Natural and synthetic estrogenic compositionswhich can be used according to the invention described herein includenatural estrogenic hormones and congeners, including but not limited toestradiol, estradiol benzoate, estradiol cypionate, estradiol valerate,estrone, diethylstilbestrol, piperazine estrone sulfate, ethinylestradiol, mestranol, polyestradiol phosphate, estriol, estriolhemisuccinate, quinestrol, estropipate, pinestrol, estrone potassiumsulfate, and tibolone. Equine estrogens, such as equilelinin,equilelinin sulfate, and estetrol, and synthetic steroids combiningestrogenic, androgenic, and progestogenic properties such as tibolonemay also be employed.

Typical dose ranges for estrogenic compounds depend on the compound andon the characteristics of the patient. For an adult human female patienttreated with a transdermal 17-β-estradiol preparation, a typical doserange is one that maintains a serum level of estradiol of about 25 pg/mLto about 140 pg/mL, more preferably between about 30 pg/mL to about 50pg/mL. A specific example of a composition containing an estrogeniccompound is one comprised of a GnRH agonist and 17-β-estradiol. The twocompounds, along with other optional excipients and/or a progestinand/or an androgenic compound, are formulated for delivery intranasally.For an intranasal preparation, a preferred daily dosage range for17-β-estradiol is between about 0.15 mg and 0.6 mg.

As discussed below, the estrogenic compound is preferablyco-administered from the same delivery vehicle or via the same route asthe GnRH compound. However, delivery of the estrogenic compound can befrom a different vehicle and/or by a different route than the GnRHcompound, and some examples of such “mixed modes” of administration areprovided below.

A3. Composition Components: Androgenic Compound

The composition comprised of a GnRH compound and an estrogenic compoundcan optionally include an androgenic compound. When present in thecomposition, the androgenic compound is in an amount effective toincrease a patient's androgen level to a level not exceeding a “normal”premenopausal level, and in particular in concert with the estrogeniccomposition to maintain bone mineral density. Such “normal” androgenlevels are on the order of about 15 ng/dL to about 80 ng/dL fortestosterone.

Suitable androgenic compounds for use in the composition include but arenot limited to testosterone, androstenedione, dihydrotestosterone,testosterone propionate, testosterone enanthate, testosterone cypionate,methyltestosterone, danazol, dromostanolone propionate, ethylestrenol,methandriol, nandrolone decanoate, nandrolone phenpropionate,oxandrolone, oxymethalone, and stanozolol.

Typical dose ranges for androgenic hormones depend upon the choice ofcompound and the individual patient. For an adult human femaleadministered testosterone, typical doses are administered to provideserum levels of testosterone of from about 15 ng/dL to about 80 ng/dL,and preferably about 40 ng/dL to about 60 ng/dL. A typical daily dosecan range from between about 0.15 mg to about 1 mg. A specific exampleof a composition containing an androgenic compound is one comprised of aGnRH agonist and 17-β-estradiol and testosterone. The compounds, alongwith other optional excipients, are formulated for deliveryintranasally, and exemplary formulations are described below.

A4. Composition Components: Progestin Compound

The composition comprised of a GnRH compound and an estrogenic compound,in some embodiments, can further include a progestin. Formulations thatinclude a progestin can be administered for a limited period of time,for example on the order of 5 to 20 days, and preferably 10 to 15 daysafter each extended treatment regimen of, for example, about 4 months,more preferably greater than about 6 months, and more specifically, offrom about 4 months to about 12 months. The progestin is provided in anamount effective to minimize or eliminate the occurrence of endometrialhyperplasia by substantially reducing the possibility of endometrialhyperstimulation which may occur during prolonged treatment withestrogenic steroids without a progestin.

Suitable progestational agents (progestins) include but are not limitedto dydrogesterone, ethynodiol diacetate, hydroxyprogesterone caproate,medroxyprogesterone acetate, norethindrone, norethindrone acetate,norethynodrel, norgestrel, progesterone, and megestrol acetate. Typicaldose ranges for progestins depend upon the choice of steroid and theindividual patient. Doses are selected as adequate to produce asecretory uterine endothelium after the time interval of progestogentreatment (about 5 to about 20 contiguous days, and preferably about 10to about 15 contiguous days). The serum level of progesterone isgenerally less than about 50 ng/dL after the time interval of progestintreatment.

B. Exemplary Nasal Preparations

As noted above, the composition comprised of a GnRH compound, andestrogenic compound, and optionally an androgen and/or a progestin, ismucosally administered by contacting the composition in a suitabledosage form with mucosal tissue of the vagina, nose, rectum, or mouth.In a preferred embodiment, the composition is administered via the nasalmucosa, i.e., intranasally. The nasal mucosa provides a usefulanatomical site for systemic delivery. The nasal tissue is highlyvascularized, providing an attractive site for rapid and efficientabsorption. The adult nasal cavity has a capacity of around 20 mL, witha large surface area of approximately 180 cm² for drug absorption, duein part to the microvilli present along the psuedostratified columnarepithelial cells of the nasal mucosa.

A nasal preparation comprised of the composition described above cantake a variety of forms for administration in nasal drops, nasal spray,gel, ointment, cream, powder or suspension, using a dispenser or otherdevice as needed. A variety of dispensers and delivery vehicles areknown in the art, including single-dose ampoules, atomizers, nebulizers,pumps, nasal pads, nasal sponges, nasal capsules, and the like.

More generally, the preparation can take a solid, semi-solid, or liquidform. In the case of a solid form, the components may be mixed togetherby blending, tumble mixing, freeze-drying, solvent evaporation,co-grinding, spray-drying, and other techniques known in the art. Suchsolid state preparations preferably provide a dry, powdery compositionwith particles in the range of between about 20 to about 500 microns,more preferably from 50 to 250 microns, for administration intranasally.

A semi-solid preparation suitable for intranasal administration can takethe form of an aqueous or oil-based gel or ointment. For example, thecomponents described above can be mixed with microspheres of starch,gelatin, collagen, dextran, polylactide, polyglycolide or other similarmaterials that are capable of forming hydrophilic gels. The microspherescan be loaded with drug, and upon administration form a gel that adheresto the nasal mucosa.

In a preferred embodiment, the nasal preparation is in liquid form,which can include an aqueous solution, an aqueous suspension, an oilsolution, an oil suspension, or an emulsion, depending on thephysicochemical properties of the composition components. The liquidpreparation is administered as a nasal spray or as nasal drops, usingdevices known in the art, including nebulizers capable of deliveringselected volumes of formulations as liquid-droplet aerosols. Forexample, a commercially available spray pump with a delivery volume of50 or 100 μL is available from, for example, Valois (Congers, N.Y.) withspray tips in adult size and pediatric size. In one embodiment, thecomposition comprised of a GnRH agonist and an estrogenic compound areco-administered intranasally via an aerosol spray in a daily volume ofbetween about 30 to about 200 μL.

The liquid preparation can be produced by known procedures. For example,an aqueous preparation for nasal administration can be produced bydissolving, suspending, or emulsifying the polypeptide and the steroidcompounds in water, buffer, or other aqueous medium, or in a oleaginousbase, such as a pharmaceutically-acceptable oil like olive oil,lanoline, silicone oil, glycerine, fatty acids, and the like.

It will be appreciated that excipients necessary for formulation,stability, and/or bioavailability can be included in the preparation.Exemplary excipients include sugars (glucose, sorbitol, mannitol,sucrose), uptake enhancers (chitosan), thickening agents and stabilityenhancers (celluloses, polyvinyl pyrrolidone, starch, etc.), buffers,preservatives, and/or acids and bases to adjust the pH.

In a preferred embodiment, an absorption promoting component isincluded. Exemplary absorption promoting components include surfactantacids, such as cholic acid, glycocholic acid, taurocholic acid, andother cholic acid derivatives, chitosan and cyclodextrins. In apreferred embodiment, a cyclodextrin is included in the preparation.Cyclodextrins are cyclic oligosaccharides of α-D-gluco-pyranose and canbe formed by the catalytic cycilization of starch. Due to a lack of freerotation about the bonds connecting the glycopyranose units,cyclodextrins are toroidal or cone shaped, rather than cylindrical. Thecyclodextrins have a relatively hydrophobic central cavity and ahydrophilic outer surface. The hydrophobic cage-like structure ofcyclodextrins has the ability to entrap a variety of guest compounds toform host-guest complexes in the solid state and in solution. Thesecomplexes are often termed inclusion complexes and the guest compoundsare released from the inclusion site.

The most common cyclodextrins are α-, β-, and γ-cyclodextrin, whichconsist of six, seven, or eight glucopyranose units, respectively.Cyclodextrins containing nine, ten, eleven, twelve, and thirteenglucopyranose units are designated δ, ε-, ξ-, η-, and θ-cyclodextrin,respectively. Characteristics of α-, β-, γ-, and δ-cyclodextrin areshown in Table 1. TABLE 1 Cyclodextrin Characteristics α-cyclodextrinβ-cyclodoextrin γ-cyclodextrin δ-cyclodextrin no. of glucopyranose units6 7 8 9 molecular weight (Daltons) 972 1135 1297 1459 central cavitydiameter (Å) 4.7-5.3 6.0-6.5 7.5-8.3 10.3-11.2 water solubility 14.51.85 23.2 8.19 (at 25° C., g/100 mL)

Derivatives formed by reaction with the hydroxyl groups lining the upperand lower ridges of the toroid are readily prepared and offer a means ofmodifying the physicochemical properties of the parent cyclodextrins.The parent cyclodextrins, and in particular β-cyclodextrin, have limitedaqueous solubility. Substitution of the hydroxyl groups, even withhydrophobic moieties such as methoxy and ethoxy moieties, typicallyincreases solubility. Since each cyclodextrin hydroxyl group differs inchemical reactivity, reaction with a modifying moiety usually producesan amorphous mixture of positional and optical isomers. The aggregatesubstitution that occurs is described by a term called the degree ofsubstitution. For example, a 2-hydroxypropyl-β-cyclodextrin with adegree of substitution of five would be composed of a distribution ofisomers of 2-hydroxypropyl-β-cyclodextrin in which the average number ofhydroxypropyl groups per 2-hydroxypropyl-β-cyclodextrin molecule isfive. Degree of substitution can be determined by mass spectrometry ornuclear magnetic resonance spectroscopy. These methods do not giveinformation as to the exact location of the substituents (C1, C2, C3,etc.) or the distribution of the substituents on the cyclodextrinmolecule (mono, di, tri, poly). Theoretically, the maximum degree ofsubstitution is 18 for α-cyclodextrin, 21 for β-cyclodextrin, and 24 forγ-cyclodextrin, however, substituents with hydroxyl groups present thepossibility for additional hydroxylalkylations.

The cyclodextrin used in the present invention is preferably an α-, β-,or γ-cyclodextrin. The cyclodextrin selected for use depending on whichcyclodextrin binds the guest compounds and yields the desiredbioavailability. In a preferred embodiment, a derivative of acyclodextrin is selected, and derivatives such as hydroxypropyl,dimethyl, and trimethyl substituted cyclodextrins are contemplated, asare cyclodextrins linked with sugar molecules, sulfonated cyclodextrins,carboxylated cyclodextrins, and amino derivatives such as diethylaminocyclodextrins. In a preferred embodiment, the cyclodextrin is aβ-cyclodextrin, and in a more preferred embodiment, the cyclodextrin is2-hydroxypropyl-β-cyclodextrin. In yet another embodiment, the2-hydroxypropyl-β-cyclodextrin has a degree of substitution between 2and 8, more preferably between 4 and 8, most preferably between 5 and 8.

In a study performed in support of the invention, the solubility of thesteroids estradiol and testosterone, alone and in combination, invarying concentrations of 2-hydroxypropyl-β-cyclodextrin was determined.As described in Example 1, defined amounts of each steroid were added to1 mL of 2-hydroxypropyl-β-cyclodextrin in water. The solubility of thesteroids was determined, and the results are shown in Tables 2A and 2B.

Table 2A shows the solubility of 17-β-estradiol and testosteroneindividually in aqueous solutions of 2-hydroxypropyl-β-cyclodextrin. Thelast two columns in the Table 2A show the molar ratio of each steroid tothe cyclodextrin. The molar occupancy of 17-β-estradiol will respect to2-hydroxypropyl-β-cyclodextrin, averages approximately 0.21. The molaroccupancy of testosterone with respect to 2-hydroxypropyl-β-cyclodextrinaverages approximately 0.39. TABLE 2A Solubility of 17-β-Estradiol andTestosterone in 2-Hydroxypropyl-β-cyclodextrin Testosterone Molar RatioHPβCD* 17-β-Estradiol solubility Estradiol/ Testosterone/ mg/mLsolubility mg/mL mg/mL HPβCD HPβCD 10 0.414 0.810 0.21 0.39 40 1.6052.541 0.20 0.30 70 2.763 5.626 0.20 0.38 100 4.062 6.819 0.21 0.33 1305.494 11.649 0.21 0.43 160 6.841 13.866 0.22 0.41 190 8.379 16.522 0.220.42 220 9.330 18.604 0.21 0.40 250 11.031 21.684 0.22 0.41*2-hydroxypropyl-β-cyclodextrin

Table 2B shows the solubility of 17-β-estradiol as a first steroid andtestosterone as a second steroid in aqueous2-hydroxypropyl-β-cyclodextrin. The last three columns show the molarratios of each steroid individually in the2-hydroxypropyl-β-cyclodextrin solution and of the two steroids togetherin the solution. The data shows that the combined molar occupancy of thetwo steroids together, average approximately 0.48, is greater than themolar occupancy achieved with either steroid alone (Table 2A). TABLE 2BMolar Occupancy of 17-β-Estradiol and Testosterone in2-Hydroxypropyl-β-cyclodextrin Estradiol and Testosterone solubilityMolar Ratio mg/mL Estradiol & HPβCD* Testos- Estradiol/ Testosterone/Testosterone/ mg/mL Estradiol terone HPβCD HPβCD HPβCD 10 0.164 0.6580.08 0.31 0.40 40 0.834 2.819 0.11 0.34 0.44 70 1.562 5.073 0.11 0.350.46 100 2.157 7.113 0.11 0.34 0.45 130 3.202 10.552 0.12 0.39 0.51 1604.053 13.422 0.13 0.40 0.53 190 4.796 15.742 0.13 0.40 0.52 250 5.77419.986 0.12 0.38 0.50*2-hydroxy-propyl-β-cyclodextrin

The molar ratio data of Tables 2A and 2B are presented graphically inFIG. 1. The figure also shows the molar ratio determined in anotherstudy where estradiol was first added to the aqueous2-hydroxypropyl-β-cyclodextrin solution, followed by addition oftestosterone. The molar occupancy of the two steroids in combination issimilar, regardless of the sequence of addition of the steroids to the2-hydroxypropyl-β-cyclodextrin solution.

In another study, the solubility of 17-β-estradiol and testosterone,alone and in combination, as a function of degree of substitution of2-hydroxypropyl-β-cyclodextrin was evaluated. Solutions of2-hydroxypropyl-β-cyclodextrin with degrees of substitution of 5.5, 6.1,and 6.8 were prepared and the maximum concentration of estradiol andtestosterone that could be solubilized was determined. There was aslight trend for the 2-hydroxypropyl-β-cyclodextrin with a lower degreeof substitution to solubilize more steroid, however, the trend was notstatistically significant.

C. In vivo Studies

In studies performed in support of the invention, the GnRH compounddeslorelin and the estrogenic compound estradiol, in the form of awater-soluble complex with the cyclodextrin2-hydroxypropyl-β-cyclodextrin, were administered intranasally topatients. These studies will now be described.

In a first study, deslorelin was administered alone to determine anappropriate dose for treatment of an exemplary benign gynecologicaldisorder, uterine leiomyomata (fibroids). More specifically, the doseappropriate to control the heavy bleeding secondary to uterineleiomyomata was determined. As described in Example 3, female patientswere treated with deslorelin administered daily via intranasal delivery,at a dose of 0.5 mg, 1.0 mg or 2.0 mg. The compound was administeredusing a commercially available nasal sprayer that delivered a 50 μLspray volume. The daily dose was administered by application of 50 μL toeach nostril once per day, for a total daily volume of 100 μL. Duringthe 12 week treatment period the patients kept daily bleeding calendarsand underwent clinical assessments at scheduled intervals. Clinicalassessments included grading of nasal irritation (Table 5A),determination of uterine size (Table 5B), and serum hormone levels(Table 5C). This clinical data are presented in the indicated tables inExample 3, along with the bleeding scores (Table 5D).

With respect to nasal irritation, the data (Table 5A in Example 3)indicates that subjects experience none or slight irritation at doses of0.5 mg and 1 mg. Some of the subjects treated with a deslorelin dose of2 mg experienced more frequent irritation.

Table 5B in Example 3 also shows a reduction in uterine volume(calculated from ultrasound determined dimensions), with the reductiondirectly correlating to dose.

Serum levels of estradiol, progesterone, and testosterone are shown inTable 5C in Example 3. Reduction in estradiol levels was progressive anddose dependent over the 12 week period. Progesterone levels followed asimilar pattern with progressive suppression with higher dose and longertime. Testosterone levels were also similar.

With respect to bleeding scores (Table 5D in Example 3), the patientstreated with the GnRH compound had a marked improvement in theirbleeding score at the end of the 12 week study.

In summary, all tested doses were partially or completely effective withreduction of bleeding, associated pain, and uterine size. This effectcorrelates with a reduction of estrogen and progesterone. Based on thebleeding scores, a 1 mg dose appears to offer a slight advantagecompared with the 0.5 mg dose. Uterine size and rates of change ofestrogen and testosterone levels are clearly dose related over the rangestudied.

Another study performed in support of the invention is described inExample 4. In this study, the GnRH compound deslorelin, estradiol, andtestosterone were co-administered as an “all-in-one” nasal spray tooophorectomized women. Each woman was treated with 50 μL of a nasalspray preparation, prepared as described in Example 2, delivered as asingle dose on two occasions separated by one week. The 50 μL dosedelivered 1 mg deslorelin, 50 μg estradiol, and 250 μg testosterone. Theestradiol and testosterone were in the form of a water-soluble complexwith cyclodextrin. Blood samples were collected prior to and afterdosing on each test day for quantitation of serum estradiol andtestosterone levels. The results are presented in FIGS. 2A and 2B.

FIG. 2A is a bar graph showing the cumulative area under the curve (AUC)from 0 to 360 minutes for serum estradiol, in pg*min/mL, for each of thesix patients. FIG. 2B is a similar graph for testosterone. The hormonelevels on day 1 and day 8, corresponding to dose 1 and dose 2, are shownas a separate bar for each patient. Comparison of the AUC of eachpatient shows that uptake of the compounds in the nasal preparation isrelatively uniform, with variations between patients likely due tovarying extent of metabolic conversion during nasal mucosa absorption.Importantly, the data also show that significant absorption of estradioland testosterone occurs in the presence of the GnRH compound.

Example 5 describes a study performed in support of the invention wherethe nasal preparation similar to that of Example 2, comprised of a GnRHcompound, estradiol, and testosterone, was administered to normalpremenopausal women. Three doses of the GnRH compound deslorelin weretested, 0.5 mg, 1.0 mg, and 2.0 mg. The nasal preparation wasadministered using a conventional metered nasal spray delivery device.The subjects received two 50 μL sprays, one in each nostril, daily forfour weeks. Blood samples were collected prior to drug administration onday 1 of the study, and then at regular intervals throughout day 1.Thereafter, blood samples were collected weekly, until day 29, whenblood was collected at scheduled intervals throughout the day. Serumdeslorelin, estradiol, testosterone, and progesterone were quantified,and the results are shown in Tables 6A through 6C.

The degree of induced ovarian suppression is evident from the serumestradiol and progesterone levels (Tables 6A-6C). Serum levels ofestradiol on day 29 varied from 14 pg/mL to 103 pg/mL. Progesteronelevels were generally less than 80 ng/dL during the treatment intervalindicating that women were anovulatory during the treatment.

In one embodiment, the intranasal dose of the estrogenic compound, andthe optional androgen if present, achieve a transient serum leveloutside the serum estradiol level of between about 25 pg/mL to about 140pg/mL that is typically reported in the literature with a 50 μg/daytransdermal patch. Although the hormonal serum levels resulting fromintranasal delivery of the hormone(s) are transiently outside this rangea similar beneficial effect is achieved. That is, the biologicaleffect(s) resulting from intranasal delivery of an estrogenic compound,and the optional androgen, is similar to the biological effectassociated with a serum estradiol level of between about 25 pg/mL toabout 140 pg/mL even though the actual transient serum level may beoutside this range. Thus, in one embodiment, the invention contemplatesadministration of an estrogenic compound and an optional androgen in anamount sufficient to achieve the beneficial biological effects that areassociated with a steady serum estradiol level of between about 25 pg/mLto about 140 pg/mL, more preferably between about 30 pg/mL to about 100pg/mL, most preferably, between about 30 pg/mL to about 50 pg/mL. Inintranasal formulations where the optional androgen is present, thetransient androgen serum blood level achieved may be lower or higherthan that typically obtained by other routes of administration. However,the beneficial effects achieved by intranasal administration are similarto those obtained from a steady serum testosterone level of betweenabout 20 ng/dL to about 80 ng/dL, more preferably between about 40 ng/dLto about 60 ng/dL.

A comparison of the total area under concentration-time curves (AUC) oraverage concentrations of serum estradiol (or testosterone) in subjectstreated with intranasal estradiol (or testosterone) and patients treatedwith estradiol (or testosterone) by another route, such as oral,provides a basis for determining the biological equivalency of differentroutes of administration. Where the AUCs or average concentrations aresimilar, despite different routes of administration or differentconcentration-time profiles, the biological effect achieved is oftensimilar. Thus, in one embodiment, the invention contemplates achievingby intranasal administration of the disclosed composition an averageserum estradiol concentration over 24 hours of between about 25 pg/mLand about 50 pg/mL. In nasal preparations containing the optionalandrogen testosterone, the invention contemplates achieving an averageserum testosterone concentration over 24 hours of between about 15 ng/dLand about 40 ng/dL.

In a study performed in support of the invention, the averageconcentration over 24 hours resulting from transdermal administration ofestradiol and from intranasal administration of estradiol were compared.As described in Example 6, test subjects received estradioltransdermally or intranasally. Transdermal estradiol was administeredusing a Noven Vivelle® or a Noven Vivelle-dot® transdermal patch, bothat dosages of 50 μg/day. The subjects treated intranasally received 350μg estradiol in a liquid spray delivered once per day. Averageconcentrations for each of the patient populations were determined fromblood samples, and the results are summarized in Table 7 below inExample 6.

The average serum estradiol concentration over 24 hours for womenreceiving estradiol transdermally from the Vivelle® patch was 34.4 pg/mLand from the Vivelle-dot® patch was 36.8 pg/mL. The average estradiolconcentration for women treated with intranasal estradiol was 37.8pg/mL. This study shows that an estrogenic compound administered as anintranasal bolus achieves a 24 hour average serum concentrationcomparable to that achieved by transdermal administration. Thus, in oneembodiment the invention provides a 24 hour average serum concentrationof estradiol from an intranasal bolus dose of estradiol that is within(plus or minus) about 10% of the 24 hour average estradiol serumconcentration achieved from transdemal estradiol administration. Thatis, the 24 hour average estradiol serum concentrated from intranasalbolus administration of estradiol is at least about 90% of the 24 houraverage estradiol serum concentration from transdermal administration ofestradiol. The result was surprising since heretofore it was unknown (i)if a bolus dose would achieve efficacious blood concentration and (ii)if a bolus dose would achieve a concentration comparable to that ofcontrolled-release transdermal dose. The data shows that an intranasalbolus dose of an estrogenic compound achieves a therapeutic bloodconcentration, and that the concentration is comparable, i.e., is withinat least about 10%, to that achieved by transdermal administration ofthe estrogenic compound.

In another study, five women were treated with intranasal testosterone.In this study , 250 μg testosterone was formulated into a nasalpreparation also containing deslorelin and estradiol (Example 2). Theformulation was administered initially on day 1 of the study, and thenagain one week later on day 8 of the study. The average serumconcentrations over 24 hours are shown in Table 3. TABLE 3 Testosterone:Average Concentration over 24 Hours after Intranasal Administration of250 μg Testosterone (average concentration Subject Number Treatment Dayover 24 hours, ng/dL) 1 Day 1 26.7 Day 8 24.1 2 Day 1 26.7 Day 8 29.9 3Day 1 19.0 Day 8 17.1 4 Day 1 18.8 Day 8 16.5 5 Day 1 19.1 Day 8 16.2Average of both observations in 21.4 each of the 5 subjects

The average concentration over 24 hours for both doses in the fivesubjects was 21.4 ng/dL. This concentration is comparable to literaturereported values achieved from transdermal administration oftestosterone. For example, in Javanbakht et al. (J. of ClinicalEndocnnology and Metabolism, 85(7): 2935, 2000) women wearing atransdermal testosterone patch for 96 hours that delivered 300 μg/dayhad an average serum concentration of 15.8 ng/dL. Thus, the nasalformulation of the present invention, i.e., a bolus dose oftestosterone, provides a similar area under the curve as a slow-releasetransdermal formulation, as evidenced by similar average concentrationvalues.

Example 7 describes a study performed in support of the invention wherethe efficacy and biologic equivalence between nasal spray add-backestradiol and transdermal estradiol add-back were evaluated. Women withendometriosis treated with intranasal deslorelin (GnRH compound) wereassigned to one of three methods of estradiol add-back: (1) 50 μg/dayestradiol transdermal patch, (2) 300 μg/day intranasal estradiol, or (3)300 μg/day intranasal estradiol with 275 μg/day intranasal testosterone.Treatment efficacy was measured by evaluating the decrease inendometriosis using a standard scoring system that takes into account 3symptoms (pelvic pain, dysmenorrhea, and dyspareunia) and two signs(pelvic tenderness and pelvic induration) of endometrosis. The resultsare shown in Table 8A in Example 7 as the composite score physicalsymptoms and signs score (CPSSS) taken as the sum of the scores for eachindividual symptom or sign (0 to 3 with 0 being not present and 3 beingthe most severe). The nasal spray preparations with at least 90% of theestradiol average serum concentration (AUC) of the transdermal patch(Table 7) were more effective than the transdermal patch.

Loss of bone mineral density (BMD) is a known side effect of treatmentwith GnRH compounds. Thus, BMD of the lumbar spine of the test subjectsin Example 7 was obtained by dual-energy X-ray absorptiometry (DEXA)prior to and after six months of treatment. The results are shown inTable 8B in Example 7 as the ratio of BMD at the six month time point(BMD_(6mo).) to the BMD prior to treatment (BMD_(baseline)). BMD ofsubjects treated with deslorelin alone in another study (Example 8) isalso shown in Table 8B for comparison. A reduction in loss of BMD byaddition of estradiol to deslorelin is apparent, since all subjectstreated with estradiol had reduced bone loss. The data also shows thatestradiol add-back in the form of an intranasal bolus is at least aseffective, and in fact slightly more effective, in preventing loss ofBMD than a transdermal estradiol add-back, as observed by comparing theBMD ratios for women receiving estradiol transdermally(BMD_(6mo)/BMD_(baseline)=0.978) and for women receiving estradiolintranasally (BMD_(6mo)/BMD_(baseline)=0.996).

The data in Tables 8A-8B demonstrate that the estradiol added-back inthe form of an intranasal bolus dose to deslorelin resulted in asignificant decrease in endometriosis symptoms and at the same timereduced the loss of BMD. A nasal spray preparation comprised of a GnRHcompound and an estrogenic compound, where the preparation has an 24hour average estrogenic compound serum concentration (AUC) within 10% ofthe 24 hour average transdermal estrogenic compound serum concentration(Table 7), was at least as effective, and preferably more effective, inpreventing loss of BMD than an add-back estrogenic compound in the formof a transdermal patch.

The incidence of endometrial hyperplasia resulting fromco-administration of a GnRH compound and unopposed estradiol wasevaluated in a study described in Example 8. One-hundred twelvepremenopausal women participated in a year long study where deslorelinwas administered daily as a nasal spray and estradiol was administeredin the form of a transdermal patch. At the end of the year endometrialbiopsies were evaluated for hyperplasia. The results, which are shown inTable 9C (see Example 8 below), show that the incidence of simplehyperplasia for untreated subjects (Arm 1, placebo/placebo) was 2.2%.The incidence of simple hyperplasia for subjects treated with intranasaldeslorelin and transdermal estradiol (Arms 3, 4 and 5) was 0% (Arms 3and 5) or 4.2% (Arm 4). The incidence in combined Arms 4 and 5 was 2.0%.These data show that delivery of a GnRH compound with unopposed estrogen(that is, estrogen in the absence of a progestogen) resulted in littlerisk of endometrial hyperplasia, with the risk no greater than that ofthe women in the untreated population. The patients in Arm 2 of thestudy, where estradiol was absent for the first six months of the studyand then added for the final six months, had a 16.7% incidence ofhyperplasia.

In a similar study, described in Example 9, twenty women were treatedwith deslorelin and unopposed estradiol. The women were divided intotreatment groups to receive intranasal deslorelin and intranasalestradiol (Arm 1), intranasal deslorelin and transdermal estradiol (Arm2), or intranasal deslorelin and intranasal estradiol plus testosterone(Arm 3). At the end of the 6 month treatment period, the endometrialresponse was evaluated by biopsy.

The results of the biopsy are shown in Table 10 presented below inExample 9. The endometrial tissue was proliferative in the 16 evaluablebiopsies, and there was no evidence of endometrial hyperplasia in any ofthese evaluable subjects after six months of treatment.

The studies described in Examples 8 and 9 show that women administered aGnRH compound and an estrogen, and optionally an androgen, bynon-gastrointestinal routes, and preferably intranasally, are not atincreased risk of endometrial hyperplasia. The data show that a GnRHcompound and an estrogenic compound can be administered to premenopausalwomen with no increased risk of developing simple endometrialhyperplasia. This observation is unexpected since typically 30% ofpostmenopausal women treated with unopposed estrogen (0.625 mg dose)develop simple hyperplasia (Gefland, M. et al. Obstetrics & Gynecology,74:398 (1989); JAMA, 275(5):370 (1996); Clisham, P. et al., Obstetrics &Gynecology, 79:196 (1992)). Premenopausal women treated with a GnRHcompound and an estrogen were expected to be similar to postmenopausalwomen, since the GnRH compound reduces serum estradiol and serumprogesterone levels. However, the data clearly demonstrated thatpremenopausal women treated with a GnRH compound and an estrogeniccompound for an extended time period (e.g., up to one year) had noincreased incidence of simple hyperplasia. Thus, addition of a progestinto the treatment regimen of a GnRH compound and an estrogenic compoundin premenopausal women was not and is not needed to protect againstsimple endometrial hyperplasia. This observation is particularly seen inwomen who have no prior estrogen deprivation (compare Arms 3, 4, 5 withArm 2 in the study describe in Example 5). The studies further suggestthat such treatment can continue for a period of about 6 to 12 months orlonger with no significant risk of developing simple hyperplasia.

Based on these studies, the phrase “no significant risk” as used hereinintends that fewer than about 10%, and more preferably less than 5%,still more preferably less than about 2% of premenopausal women treatedwith a GnRH compound and an estrogenic compound are at risk ofdeveloping simple endometrial hyperplasia. In summary, the studies showthat treatment of benign gynecological disorders with a GnRH compoundand an estrogenic compound and, optionally, an androgenic compound, orcontraception with the two or three compounds, need not be accompaniedby treatment with a progestin on a regular or periodic basis in order toprotect against simple endometrial hyperplasia or cancer.

From the foregoing, it can be seen how various objects and features ofthe invention are met. Contraception and/or treatment of benigngynecological disorders by intranasal delivery of a GnRH compound and anestrogenic compound effectively suppresses ovarian estrogen andprogesterone production. The nasal preparations described herein containthe estrogenic compound in the form of a water-soluble complex with awater-soluble cyclodextrin. In some embodiments, a second steroid, suchas testosterone or a progestin, is included. Absorption of the GnRHcompound in the presence of the steroids is adequate to achievesuppression of ovarian estrogen and progesterone production. Further,the nasal formulations tested were non-irritating to the test subjects.

The studies described herein also showed that the nasal preparation,when administered to premenopausal women not receiving exogenouslysupplied progesterone, did not increase the risk of simple endometrialhyperplasia, relative to women receiving placebo. The studies suggestthat such treatment can continue for a period of 6 to 12 months orlonger with no significant risk of simple endometrial hyperplasia.

Ill. EXAMPLES

The following examples further illustrate the invention described hereinand are in no way intended to limit the scope of the invention.

Example 1 Solubility of Estradiol and Testosterone in Cyclodextrin/Water

The solubility of 17-β-estradiol and testosterone in varyingconcentrations of 2-hydroxypropyl-β-cyclodextrin (MW 1380 g/mole; 5.5degree of substitution) was determined as follows. 10 ng 17-β-estradiol(MW 272.39 g/mole) was added to 1 mL of 2-hydroxypropyl-β-cyclodextrinin water, the 2-hydroxypropyl-β-cyclodextrin concentration ranging from10 to 250 ng/mL. In a second series of vials, 20 ng of testosterone (MW288.43 g/mole) was added to 1 mL of 2-hydroxypropyl-β-cyclodextrin inwater, the 2-hydroxypropyl-β-cyclodextrin concentration ranging from 10to 250 ng/mL. In a third set of vials 10 ng 17-β-estradiol and 20 ngtestosterone were added to 1 mL of 2-hydroxypropyl-β-cyclodextrin inwater, the 2-hydroxypropyl-β-cyclodextrin concentration ranging from 10to 250 ng/mL. The vials were mixed at room temperature for about 1 hour.Aliquots were taken from the supematant of each vial and assayed forsteroid concentration. The results are shown in Tables 2A and 2B.

Example 2 Preparation of Intranasal Formulation

2-Hydroxypropyl-β-cyclodextrin was added to water at a concentration of240 mg/mL and stirred until dissolved. 17-β-Estradiol was then added tothe water-cyclodextrin solution at a concentration of 1.0 mg/mL. Themixture was stirred until dissolution. Testosterone at a concentrationof 5.0 mg/mL was then added, and after stirring to dissolutionbenzalkonium chloride (0.1 mg/mL), ethylene diamine tetra acetic acid(EDTA; 1 mg/mL), and sorbitol (61.6 mg/mL) were added. The mixture wasstirred. Then, the GnRH compound deslorelin acetate was added at aconcentration of 20 mg/mL with stirring. The volume was brought to thefinal desired volume and the pH was adjusted as needed. Table 4summarizes the preparation components, concentrations, and dosages per50 μL. TABLE 4 Components in Exemplary Nasal Preparation ConcentrationComponent (mg/mL) Dose per 50 μL deslorelin acetate 20 1.0 mg17-β-estradiol 1.0 50 μg Testosterone 5.0 250 μg2-hydroxypropyl-β-cyclodextrin 240 12 mg benzalkonium chloride 0.1 5 μgEDTA 1.0 50 μg Sorbitol 61.6 3.1 mg water, USP as required

Example 3 Intranasal Administration of Deslorelin to Premenopausal Womenwith Uterine Leiomyomata

A 12 week study was performed to establish an effective dose ofdeslorelin for controlling heavy bleeding secondary to uterineleiomyomata. Forty-one premenopausal women completed the study and areidentified as Subject Nos. 1 through 41. The women were divided intotest groups for treatment with intranasal deslorelin as follows: Group 1Subject Nos. 1-6 placebo, 0 mg deslorelin Group 2 Subject Nos. 7-21 0.5mg deslorelin, once per day Group 3 Subject Nos. 22-34 1.0 mgdeslorelin, once per day Group 4 Subject Nos. 35-41 2.0 mg deslorelin,once per dayThe intranasal preparation consisted of deslorelin at the indicatedconcentration along with sorbitol (61.6 mg/mL), benzalkonium chloride(0.1 mg/mL), and water.

The average age of the women was 42.3 years, with similar distributionamong groups. For one complete menstrual cycle prior to treatment, eachwoman completed a daily bleeding calendar. Eligible subjects were thentreated with deslorelin at the assigned dosage once per day byintranasal application. During the 12 weeks of daily intranasaladministration, each woman kept a daily bleeding calendar, completedquality of life questionnaires, and underwent clinical assessment andlaboratory testing. The subjects were tracked for 6 weeks post-treatmentfor further assessment and to document time to recovery of menses afterlast drug treatment day. Clinical assessments included grading of nasalirritation (Table 5A), determination of uterine size (Table 5B), andserum hormone levels (Table 5C). The bleeding scores are presented inTable 5D. TABLE 5A Nasal Irritation Number of Study Subjects Deslorelin(mg/day) 0 mg 0.5 mg 1 mg 2 mg Baseline None 11 11 13 9 Slight 2 3 0 2Moderate 0 0 1 3 Quite a bit 0 0 0 0 Extreme 0 0 0 0 Total 13 14 14 14End of None 13 11 13 12 Week 4 Slight 0 2 0 1 Moderate 0 1 1 0 Quite abit 0 0 0 1 Extreme 0 0 0 0 Total 13 14 14 14 End of None 13 11 13 11Week 8 Slight 0 1 1 3 Moderate 0 1 0 0 Quite a bit 0 1 0 0 Extreme 0 0 00 Total 13 14 14 14 End of None 13 11 13 8 Week 12 Slight 0 2 0 5Moderate 0 0 0 1 Quite a bit 0 1 1 0 Extreme 0 0 0 0 Total 13 14 14 14

TABLE 5B Uterine Size Deslorelin (mg/day) Mean Difference* 0 mg +11.60.5 mg   −109.6 1 mg −63.6 2 mg −246.5*End of week 12 minus baseline uterine volume (cm³)

TABLE 5C Hormone Levels Estradiol Progesterone Testosterone (pg/mL)(ng/dL) (ng/dL) Baseline 0 mg 164 560 32 0.5 mg 173 641 24 1 mg 123 48026 2 mg 184 337 34 End of Week 4 0 mg 141 121 27 0.5 mg 47 40 19 1 mg 5112 22 2 mg 14 12 16 End of Week 8 0 mg 134 142 31 0.5 mg 79 30 22 1 mg95 25 20 2 mg 31 14 20 End of Week 12 0 mg 152 382 29 0.5 mg 66 37 23 1mg 30 10 16 2 mg 30 13 20

TABLE 5D Bleeding Score* Deslorelin (mg/day) 0 mg 0.5 mg 1 mg 2 mgBaseline Score 10.2 10.9 10.8 14.1 Weeks 1-4 9.5 6.6 7.8 6.6 Weeks 5-88.4 3.5 0.4 1.2 Weeks 9-12 5.2 2.6 1.7 0.6*Bleeding scores were calculated from the sum of daily diary entries forthe 28 day interval prior to the reporting period. No bleedingthroughout the interval was a score of 0, ‘normal’ menstrual flow was ascore of 5, and menorrhagia was a score of 10 or greater.

Example 4 Intranasal Administration of GnRH compound, an Estrogen, andan Androgen to Oophorectomized Women

Six volunteer women with prior oophorectomies and not presently onhormone replacement therapy were recruited. Each woman was treated with50 μL of a nasal spray preparation, prepared as described in Example 2,on two occasions separated by one week. The 50 μL dose delivered 1 mgdeslorelin, 50 g 17-β-estradiol, and 250 μg testosterone. Blood sampleswere collected 20 minutes and 10 minutes prior to dosing on day 1 and onday 8, and then at the following intervals after dosing on each day: 10,20, 30, 40, 60, 90, 120, 180, 240, 360, and 1440 minutes. Serumestradiol and testosterone levels were determined from the samples, andthe baseline corrected cumulative area under the curve from 0 to 360minutes for each patient for each dose are presented in FIGS. 2A-2B.

Example 5 Intranasal Administration of Deslorelin, Estradiol, andTestosterone to Premenopausal Women

Nine premenopausal women, ages 20-45 years, were recruited and randomlydivided into three test groups for a 29 day study. The patients in Group1, Group 2, and Group 3 were treated with the intranasal preparationsimilar to that described in Example 2 but with deslorelin acetateconcentrations of 5 mg/mL (Group 1), 10 mg/mL (Group 2), or20 mg/mL(Group 3). The single daily intranasal administration consisted of a 100μL dose delivered using a metered nasal spray device as two 50 μLsprays, one in each nostril.

An indwelling intravenous catheter was inserted in an arm vein forwithdrawal of blood samples prior to drug administration and at definedintervals post administration (study day 1) of 40, 120, 240, and 480minutes. Thereafter, weekly blood samples (study days 8, 15, and 22)were collected for determination of serum estradiol, progesterone,testosterone, and deslorelin levels. On study day 29 postadministration, blood samples were drawn according to the same regimenas on study day 1. After collection, all blood samples were allowed toclot at room temperature, then refrigerated. Within 24 hours ofcollection, serum was separated and stored frozen at −5° C. untilassayed.

Serum levels of estradiol, testosterone, and progesterone werequantitated by sensitive and specific radioimmunoassay methods(Stanczyk, F. Z. et al., Am. J Obstet. Gynecol., 159(6):1540 (1988);Scott et al., Am. J Obstet. Gynecol., 130(7):817 (1978)). Prior to assayof the steroid hormones, serum was extracted with ethyl acetate:hexane(1:1) and for the testosterone assay further purified via Celite™ columnchromatography, with 40% toluene to elute the testosterone. Procedurallosses were followed by addition of 1000 dpm of the appropriatetritiated internal standard. The sensitivities of the estradiol,testosterone, and progesterone assays were 8 pg/mL, 4 ng/dL, and 10ng/dL, respectively. Assay accuracy was demonstrated by observedparallelism between standard curves and serially diluted serum withrespect to each hormone. Intra- and inter-assay coefficients ofvariation were 5 to 10% and 10 to 15%, respectively. Specificity of theassays was enhanced by eliminating interfering metabolites withextraction and/or chromatography and through the use of highly specificantisera.

The results of all hormone analyses are presented for each subject inTables 6A through 6C. TABLE 6A Hormone Levels for Patients Treated with0.5 mg/mL Deslorelin Study Study Estradiol Testosterone ProgesteroneSubject* Day Time pg/mL ng/dL ng/dL #1 1 0 61 31 110 40 91 142 120 240480 8 159 29 90 15 300 28 100 22 10 15 40 29 0 14 13 100 40 57 137 120240 480 42 1260 #2 1 0 33 18 100 40 42 43 120 240 480 8 130 25 50 15 16025 40 22 39 19 50 29 0 52 18 60 40 50 20 120 240 480 42 40 #3 1 0 54 2190 40 128 190 120 240 480 8 85 39 110 15 98 36 22 37 22 40 29 0 103 8670 40 112 142 120 240 480 42*Treated with the nasal preparation of Example 2 having 0.5 mg/mLdeslorelin acetate.

TABLE 6B Hormone Levels for Patients Treated with 1.0 mg/mL DeslorelinStudy Study Estradiol Testosterone Progesterone Subject* Day Time pg/mLng/dL ng/dL #4 1 0 73 21 40 40 98 81 120 240 480 8 167 31 50 15 91 33 5022 86 24 50 29 0 40 23 60 40 126 144 120 240 480 42 #5 1 0 73 21 220 40227 250 120 240 480 8 26 19 50 15 27 23 30 22 132 37 50 29 0 45 25 40 4098 102 120 240 480 42 #6 1 0 51 20 60 40 181 174 120 240 480 8 45 26 8015 18 20 70 22 48 16 60 29 0 29 20 100 40 89 98 120 240 480 42*Treated with the nasal preparation of Example 2 having 1.0 mg/mLdeslorelin acetate.

TABLE 6C Hormone Levels for Patients Treated with 2.0 mg/mL DeslorelinStudy Study Estradiol Testosterone Progesterone Subject* Day Time pg/mLng/dL ng/dL #7 1 0 32 14 90 40 141 149 120 240 480 8 21 13 50 15 16 1460 22 12 9 40 29 0 23 10 70 40 40 24 120 240 480 42 #8 1 0 61 15 80 40191 244 120 240 480 8 149 30 50 15 302 39 70 22 22 22 150 29 0 90 25 7040 117 88 120 240 480 42 #9 1 0 37 21 30 40 152 241 120 240 480 8 158110 20 15 18 18 20 22 24 15 10 29 0 29 14 20 40 143 132 120 240 480 42*Treated with the nasal preparation of Example 2 having 2.0 mg/mldeslorelin acetate.

Example 6 Comparison of Intranasally and Transdermally DeliveredEstradiol

Naturally postmenopausal or surgically postmenopausal females (n=63)were recruited for the study. Thirty women were selected for treatmentwith transdermal estradiol from a Noven Vivelle® 50 μg/day patch. Thirtywomen were treated with transdermal estradiol from a Noven Vivelle-dot®50 μg/day patch. The remaining three women were treated with a singlebolus, 100 μL volume, nasal spray containing 350 μg 17-β-estradiol per100 μL bolus. The nasal formulation in addition to estradiol wascomprised of sorbitol (61.6 mg/mL), EDTA (1.0 mg/mL), benzalkoniumchloride (0.1 mg/mL), and 2-hydroxypropyl-β-cyclodextrin (100 mg/mL).

Blood samples were drawn at defined intervals for analysis of serumestradiol levels. The average concentration over 24 hours as pg/mL wasdetermined and the results are shown in Table 7. TABLE 7 AverageEstradiol Concentration Over 24 Hours (pg/mL) after Treatment withTransdermal and Intranasal Estradiol Average Concentration overEstradiol Dosage Form 24 Hours (pg/mL) transdermal, Vivelle ® 50 μg/daypatch 34.4 transdermal, Vivelle-dot ® 50 μg/day patch 36.8 intranasal,350 μg/spray 37.8

Example 7 Intranasal Delivery of GnRH Compound with Transdermal orIntranasal Co-Administration of Estradiol

Women with endometriosis treated with intranasal deslorelin (GnRHcompound) were assigned to one of three methods of add-back: 1) 50μg/day estradiol transdermal patch, 2) 300 μg/day intranasal estradiol,or 3) 300 μg/day intranasal estradiol with 275 pg/day intranasaltestosterone. Treatment efficacy was measured by evaluating the decreasein endometriosis symptoms associated with treatment. Evaluated weresymptoms and signs of endometriosis using a standard scoring system thattakes into account 3 symptoms (pelvic pain, dysmenorrhea, anddyspareunia) and two signs (pelvic tenderness and pelvic induration).The composite score physical symptoms and signs score (CPSSS) is the sumof the scores for each individual symptom or sign (0 to 3 with 0 beingnot present and 3 being the most severe). Shown in Table 8A is thechange in CPSSS following 3 and 6 months of treatment. TABLE 8A CPSSSafter 3 and 6 Months of Drug Treatment Baseline No. of CPSSS Month 3Month 6 subjects Deslorelin + Transdermal 7.4 4.0 3.8 5 EstradiolDeslorelin + Intranasal 8.1 2.3 3.4 7 Estradiol Deslorelin + Intranasal6.8 1.6 3.1 8 Estradiol + Testosterone

Bone mineral density (BMD) of the lumbar spine was obtained bydual-energy X-ray absorptiometry (DEXA) prior to and after six months ofdrug treatment. BMD changes are shown in Table 8B as the ratio of the 6month value compared to the baseline value. BMD of subjects treated withdeslorelin alone (Example 8) was 0.971 of the baseline value. TABLE 8BEffect of Add-back Estradiol on the Ratio of 6 Month BMD to Baseline BMDMean Ratio No. of (BMD_(6 mo.)/BMD_(baseline)) subjects Deslorelin*0.971 36 Deslorelin + Transdermal Estradiol 0.978 5 Deslorelin +Intranasal Estradiol 0.996 7 Deslorelin + Intranasal Estradiol + 0.999 8Testosterone*From study described in Example 8.

Example 8 Intranasal Delivery of GnRH Compound with TransdermalCo-Administration of Estradiol

Example 8 describes an intranasally delivered GnRH compound withco-administration of estradiol. Premenopausal females (n=265) withuterine fibroids were recruited for participation in a 12 month doubleblind study. The women were randomly assigned to treatment in one of thefollowing five study arms:

Arm 1 placebo/placebo

Arm 2 deslorelin, intranasal/placebo for 6 months; then crossed over toarm 5

Arm 3 deslorelin, intranasal/25 μg estradiol, transdermal

Arm 4 deslorelin, intranasal/50 μg estradiol, transdermal

Arm 5 deslorelin, intranasal/75 μg estradiol, transdermal

Deslorelin at a daily dose of 1 mg was administered intranasally using aconventional metered spray delivery device. The intranasal preparationwas a 100 μL daily nasal spray containing 1.0 mg deslorelin. Estradiolwas administered transdermally using a commercially availabletwice-weekly patch that delivered either 25 μg estradiol or 50 μgestradiol per day. Subjects in Arm 5 wore two patches, one at eachdosage, to achieve the 75 μg dose.

Women with uterine fibroids often experience very heavy bleeding(menorrhagia) and uterine enlargement leading to pelvic pain andpressure symptoms. Clinical benefit was assessed by measuring changes inbleeding score (percent of subjects with reduction of bleeding into thenormal range) and uterine volume. Shown in Table 9A are the percent ofsubjects responding (reduction of bleeding into the normal range) after3 and 6 months of treatment. The percent of subjects having a responseis significantly greater in the active treatment groups compared to theplacebo group. Table 9B shows the change in uterine volume (expressed asproportion of initial volume) associated with treatment. The placebogroup had an increase in uterine volume and active treatment resulted indecreased uterine volume. TABLE 9A Percent of Subjects with Reduction ofBleeding into the Normal Range 3 Months 6 Months No. of % with No. of %with Subjects reduction subjects reduction Placebo 79 10 70 17Deslorelin 36 69 36 92 Deslorelin + 25 μg/day 32 75 27 85 transdermalestradiol Deslorelin + 50 μg/day 37 73 35 66 transdermal estradiolDeslorelin + 75 μg/day 42 81 38 68 transdermal estradiol

TABLE 9B Proportion of Uterine Volume: Volume after 6 Months ofTreatment Compared to Baseline 6 Months No. of Proportion of subjectsInitial Volume Placebo 60 1.238 Deslorelin 33 0.675 Deslorelin + 25μg/day transdermal estradiol 24 0.805 Deslorelin + 50 μg/day transdermalestradiol 31 0.789 Deslorelin + 75 μg/day transdermal estradiol 37 0.910

Safety was assessed by examining endometrial tissue for evidence ofhyperplasia. At the end of the 12 month treatment period an endometrialbiopsy was taken for analysis of the endometrial morphology. The resultsare shown in Table 9C. TABLE 9C Incidence of Simple Hyperplasia fromEndometrial Biopsy Arm 1 2 3 4 5 4&5 No. of subjects 1 2 0 1 0 1 withsimple hyperplasia Total no. of 45 12 6 24 25 49 subjects studiedProportion of 0.022 0.167 0.000 0.042 0.000 0.020 subjects with simplehyperplasia

Example 9 Intranasal Delivery of GnRH Compound with Co-administration ofTransdermal or Intranasal Estradiol±Testosterone

Twenty premenopausal women with endometriosis were recruited andrandomly assigned for treatment as follows: Arm 1 deslorelin,intranasal/estradiol, transdermal n = 5 Arm 2 deslorelin, and estradiol,intranasal n = 7 Arm 3 deslorelin, estradiol, and testosterone,intranasal n = 8

The Arm 1 intranasal formulation contained 1 mg deslorelin; estradiolwas delivered transdermally from a twice-weekly commercially-available50 μg/day estradiol patch. The intranasal formulations used in Arms 2and 3 contained 1 mg deslorelin and 300 μg estradiol (Arm 2), andadditionally 275 μg testosterone (Arm 3), formulated in a similar manneras that described in Example 2.

After the six month treatment period the incidence of endometrialhyperplasia was evaluated by biopsy in 20 subjects. The results areshown in Table 10. TABLE 10 Endometrial Response Insufficient Re-Hyperplasia Proliferative Atrophic Tissue fused Baseline 0 17 0 3 0Month 6 0 16 0 3 1

Although the invention has been described with respect to particularembodiments, it will be apparent to those skilled in the art thatvarious changes and modifications can be made without departing from theinvention.

1. An intranasal drug-delivery system for use in female contraception orin the treatment of benign gynecological disorders, comprising anebulizer operable to deliver a selected volume, and contained in thenebulizer, a liquid formulation composed of (i) a liquid carrier, (ii) aGnRH compound capable of suppressing ovarian estrogen and progesteroneproduction, and (iii) an estrogenic compound capable of preventing signsand symptoms of estrogen deficiency when co-administered with the GnRHcompound, where the concentration of GnRH compound and estrogeniccompound in the formulation are effective, when administeredintranasally, to suppress ovarian function and to prevent signs andsymptoms of estrogen deficiency, without a significant increase in therisk of endometrial hyperplasia.
 2. The intranasal drug-delivery systemof claim 1, wherein said nebulizer is operable to deliver a selectedvolume between 30 and 200 μL of an aqueous formulation in the form of aliquid-droplet aerosol.
 3. The intranasal drug-delivery system of claim1, wherein said liquid formulation further comprises a water-solublecyclodextrin present in the form of a water-soluble complex with theestrogenic compound.
 4. The intranasal drug-delivery system of claim 3,wherein the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin, at aconcentration between 50 and 300 mg/mL.
 5. The intranasal drug-deliverysystem of claim 4, wherein the 2-hydroxypropyl-β-cyclodextrin has adegree of substitution between 2 and
 8. 6. The intranasal drug-deliverysystem of claim 1, wherein the GnRH compound is deslorelin, at a dosebetween 0.025 and 1.5 mg.
 7. The intranasal drug-delivery system ofclaim 1, wherein the estrogenic compound is 17β-estradiol, at a dosebetween 0.15 and 0.6 mg.
 8. The intranasal drug-delivery system of claim3, which further includes testosterone as a second or third steroid inthe form of a water-soluble complex with the cyclodextrin, and at a doseof between 0.15 and 1 mg.
 9. The intranasal drug-delivery system ofclaim 3, which further includes a progestin as a second or third steroidin the form of a water-soluble complex with the cyclodextrin.
 10. Theintranasal drug-delivery system of claim 8, wherein the estrogeniccompound and the second or third steroid have a combined molar occupancywith respect to the cyclodextrin that is greater than the molaroccupancy achievable with either steroid alone.
 11. The intranasaldrug-delivery system of claim 9, wherein the estrogenic compound and thesecond or third steroid have a combined molar occupancy with respect tothe cyclodextrin that is greater than the molar occupancy achievablewith either steroid alone.
 12. The intranasal drug-delivery system ofclaim 3, wherein the cyclodextrin is 2-hydroxypropyl-β-cyclodextrinhaving a degree of substitution between 2 and 8, and a concentrationbetween 50 and 300 mg/mL.
 13. The intranasal drug-delivery system ofclaim 1, wherein the GnRH compound is a GnRH peptide agonist.
 14. Theintranasal drug-delivery system of claim 1, wherein the GnRH compound isselected from the group consisting of deslorelin, leuprolide, nafarelin,goserelin, decapeptyl, buserelin, histrelin, gonadorelin, abarelix,cetrorelix, azaline B, and degarelix, and analogs thereof.